The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy

Article abstract of DOI:10.1016/S0968-0896(00)00331-X

RPR132331, a 2-(2-dioxanyl)imidazole, was identified as an inhibitor of tumour necrosis factor (TNF)α release from lipopolysaccharide (LPS)-stimulated human monocytes. An intensive programme of work exploring the biology, toxicity and physical chemistry of a novel series of inhibitors, derived from RPR132331, has led to the identification of RPR200765A, a development candidate for the treatment of rheumatoid arthritis (RA). RPR200765A is a potent and selective inhibitor of p38 MAP kinase (IC₅₀ = 50 nM). It inhibits LPS-stimulated TNFα release both in vitro, from human monocytes (EC₅₀ = 110 nM), and in vivo in Balb/c mice (ED₅₀ = 6 mg/kg). At oral doses between 10 and 30 mg/kg/day it reduces the incidence and progression in the rat streptococcal cell wall (SCW) arthritis model when administered in either prophylactic or therapeutic dosing regimens. The compound, which is a mesylate salt and exists as a stable monohydrate, shows good oral bioavailabiltiy (F = 50% in the rat) and excellent chemical stability. The data from the SCW disease model suggests that RPR200765A could exhibit a profile of disease modifying activity in rheumatoid arthritis (RA) patients which is not observed with current drug therapies.

Full text of DOI:10.1016/S0968-0896(00)00331-X

Bioorganic & Medicinal Chemistry 9 (2001) 537±554
The Discovery of RPR 200765A, a p38 MAP Kinase Inhibitor
Displaying a Good Oral Anti-Arthritic Ecacy
Iain M. McLay,* Frank Halley,* John E. Souness, Je€rey McKenna, Veronique Benning,
Mark Birrell, Brenda Burton, Maria Belvisi, Alan Collis, Alex Constan, Martyn Foster,
David Hele, Zaid Jayyosi, Mike Kelley, Chris Maslen, Glen Miller,
Marie-Claude Ouldelhkim, Kenneth Page, Simon Phipps, Kenneth Pollock, Barry Porter,
Andrew J. Ratcli€e, Elisabeth J. Redford, Stephen Webber, Bryan Slater,
Veronique Thybaud and Nicola Wilsher
Aventis, Dagenham Research Centre, Rainham Road South, Dagenham, Essex RM10 7XS, UK
Received 12 September 2000; accepted 30 November 2000
AbstractÐRPR132331, a 2-(2-dioxanyl)imidazole, was identi®ed as an inhibitor of tumour necrosis factor (TNF)a release from
lipopolysaccharide (LPS)-stimulated human monocytes. An intensive programme of work exploring the biology, toxicity and phy-
sical chemistry of a novel series of inhibitors, derived from RPR132331, has led to the identi®cation of RPR200765A, a develop-
ment candidate for the treatment of rheumatoid arthritis (RA). RPR200765A is a potent and selective inhibitor of p38 MAP kinase
(IC50=50 nM). It inhibits LPS-stimulated TNFa release both in vitro, from human monocytes (EC50=110 nM), and in vivo in
Balb/c mice (ED50=6 mg/kg). At oral doses between 10 and 30 mg/kg/day it reduces the incidence and progression in the rat
streptococcal cell wall (SCW) arthritis model when administered in either prophylactic or therapeutic dosing regimens. The com-
pound, which is a mesylate salt and exists as a stable monohydrate, shows good oral bioavailabiltiy (F=50% in the rat) and
excellent chemical stability. The data from the SCW disease model suggests that RPR200765A could exhibit a pro®le of disease
modifying activity in rheumatoid arthritis (RA) patients which is not observed with current drug therapies. # 2001 Elsevier Science
Ltd. All rights reserved.
Introduction
joint destruction which characterises chronic rheumatoid
arthritis.2 The release of, and responses to, TNFa and IL-
1b are regulated by a mitogen-activated protein (MAP)
kinase known as p38.3a,b Indeed, selective inhibitors of
this kinase, such as the prototypical SB203580,3a potently
suppress the release of these pro-in¯ammatory cytokines
from mononuclear phagocytes and block their actions
in a variety of in¯ammatory cells. P38 is thus an
important potential target for the discovery of novel,
disease modifying anti-RA drugs. The relevance of p38
and other kinases to in¯ammatory diseases has been
reviewed.4
Rheumatoid arthritis (RA) is a chronic debilitating dis-
ease, which is estimated to a‚ict more than 2 million
people in the United States alone.1 Although palliative
treatments (non-steroidal anti-in¯ammatory drugs) are
abundant, the current disease modifying anti-rheumatic
drugs (DMARDS) are generally poorly tolerated and, as a
result, treatment regimens are frequently dose restricted
and insucient to modify the insidious progression of the
disease, which frequently leads to physical incapacita-
tion and reduced life expectancy.
Tumour necrosis factor (TNF)a and interleukin (IL)-1b
are pro-in¯ammatory cytokines implicated as causal
agents in the onset and progression of the bone and
A series of diphenylimidazoles, typi®ed by RP66425,
had been developed by Rhone Poulenc chemists as
potent, stable and well absorbed ACAT (acyl-CoA:
cholesterol O-acyl transferase) inhibitors.5 However,
unlike SB203580, no members of this series of com-
pounds were found to inhibit release of TNFa from
human monocytes following LPS stimulation. It was
*Corresponding authors. Fax: +33-(0)155718014; e-mail: frank.halley@
aventis.com
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00331-X

538
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
Scheme 1. Preparation of the dimethylacetal 6. (i) (CH3)3SiCH2CH2OCH2Cl, NaH, DMF; (ii) BuLi, N-formylmorpholine, THF; (iii) methanol,
HC(OCH3)3, p-toluenesulphonic acid.
Scheme 2. Preparation of RPR132331. (i) 4-Toluenesulphonic acid, THF.
Scheme 3. General method A, library 1 (see Table 1 for the list of diols 26). (i) General method A: 4-toluenesulphonic acid, CH2Cl2.
postulated that the existence of a 4-pyridyl group
may be a requisite for such activity. RPR132331, a 4-
pyridyl analogue of RP66425, was duly prepared
(Scheme 2) and shown to have an EC50 in this assay of
800 nM. This paper describes the rapid optimisation of
RPR132331 to provide the development candidate
RPR200765A.
(VK19911)13 but toxicity of these compounds has not
been disclosed. Two compounds have been reported to
be in clinical phase I and II, respectively, for rheuma-
toid arthritis, SB24223514a (structure not disclosed) was
shown to be orally potent14b although no toxicity issues
were discussed and VX74515a,b was reported to be devoid
of toxicity side e€ects arising from cytochrome P 450
modulation.15c
Several groups have reported p38 inhibitors with potent
in vitro and in vivo activity. SB203580 itself has been
shown to have many interesting activities in vivo,6 but
toxicological studies on rats have shown liver weight
increases and signi®cant elevation of hepatic P450
enzymes,7 whilst in vitro studies have demonstrated
inhibitory e€ects on human cytochrome P450 enzymes.7
However, more recentanalogues from hte SB labora-
tories, SB2200258 and SB226882,9 which incorporate an
aminopyrimidine moiety, display a markedly reduced
cytochrome P450 inhibition pro®le.10 Inhibitors have also
been reported by other groups, for example, Merck
(L167307),11 R.W. Johnson (RWJ68354)12 and Vertex
The mode of binding of this class of compound to p38
has been revealed through X-ray analysis of protein-
inhibition complexes.16,17 The inhibitors bind in the
region otherwise occupied by ATP. The importance of
the 4-pyridyl is clear as the pyridine nitrogen forms a
key interaction with the main chain NH of Met109. The
4-¯uorophenyl group sits in a hydrophobic pocket
which includes the speci®city residue Thr10618,19 and
the central imidazole ring forms interactions with the
enzyme through water bridges. Substituents at position
C2 of the imidazole bind in the ATP phosphate binding
region whilsta piperidine ring atN1, as found in

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
539
Table 1. Diols (26) used with general method A, Scheme 6 for
library 1
VK19911,17 appears to form a salt bridge with Asp168.
The passage of the p38 inhibitors into clinical development
has been surprisingly slow and atthe commencementof the
work described here itappeared likely thattoxicity was an
issue with compounds of this type. Certainly at that time,
toxicity in the rat had been reported for two compounds of
similar structure to SB203580: SK&F105809 and SK&F-
86002, with the ®rst shown to be genotoxic and hepato-
carcinogenic20 and the second to induce hepatocellular
hypertrophy.21 Therefore, itwas decided, before hte
optimisation of RPR132331 was initiated, to determine
toxicity parameters that could be incorporated into the
overall optimisation process. For this reason SB203580
was evaluated in models of mutagenicity and hepato-
toxicity. In vitro, in the Chinese hamster ovary micro-
nucleus assay,22 itwas found thatSB203580 was indeed
a powerful mutagen (see Table 8), whilst when dosed to
rats at 100 and 200 mg/kg/day for 14 days the compound
was found to produce a large increase in both liver
weightand CYP 1A1 conetnt(induciton of CYP 1A1
could be associated with the development of hepato-
carcinomas in rats).23 In addition to these toxicity concerns
it was known that the series of ACAT inhibitors, from
which RPR132331 was derived, can produce adrenal
toxicity which is demonstrable in the guinea pig.24
Therefore, evaluation of mutagenicity, CYP 1A1 induc-
tion and guinea pig toxicity were all incorporated into
the optimisation process, which also involved evaluation
of cellular activity, in vivo activity, bioavailability, disease
modifying activity, selectivity and physical chemical prop-
erties. The overall objective of this work was to identify a
p38 inhibitor with in vivo disease modifying activities
similar to SB203580, but without the toxicities associated
with either SB203580 or ACAT inhibitors. Clearly the
identi®cation of p38 inhibitors, suitable for clinical eva-
luation, is a highly competitive area and, consequently,
it was decided that the evaluation of RPR132331 should
be completed within one year. To achieve this, a rigid
projectplan was designed, in which a programme of
rapid parallel synthesis was allied to a rigid screening
cascade. A four-month period was allotted in this plan
for parallel synthesis and seven libraries, derived from
seven reasonably accessible templates, were designed to
make best use of this time.
Diols (26)
Cis/trans isomers
Isolated?
NA
No
Yes
NA
NA
NA
No
Yes
No
No
Yes
Yes
NA
Yes
Yes
Yes
Chemistry
Preparation of the dimethyl acetal 6, the key inter-
mediate to all this work, from 4-(4-pyridyl)-5-(4-¯uoro-
phenyl)imidazole25 1 is shown in Scheme 1. Scheme 2
shows the preparation in one step of our lead com-
pound, RPR132331 from the intermediate 6 with 2,2-di-
methyl-1,3-propane diol 7. Details of the seven small
libraries are given in Schemes 3, 7 and 8. The library
members were carefully selected to include a wide range
of biologically acceptable groups capable of exploring
H-bonding, charge, hydrophobic and aromatic interac-
tions. Each of the seven libraries required the preparation
of a template with an appropriate synthetic handle to
allow rapid and simple compound preparation. In
addition, a molecular weight restriction of 550 and a
No
Yes

540
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
Scheme 4. Preparation of the two acids 10 and 11. (i) 4-Toluenesulphonic acid, THF; (ii) NaOH, H2O, methanol.
Scheme 5. Preparation of the two amines 16 and 17. (i) K2CO3, (CF3CO)2O, DMF; (ii) 4-toluenesulphonic acid, THF; (iii) ¯ash chromatography on
silica; (iv) K2CO3, methanol, H2O.
Scheme 6. Preparation of the two amines 24 and 25. (i) HCl (conc), methanol; (ii) 4-toluenesulphonic acid, THF; (iii) HCOONH4, methanol. Pd/C
(iv) K2CO3, (CF3CO)2O, DMF; (v) preparative HPLC; (vi) K2CO3, methanol, H2O.

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
541
separation of the isomers. The two amines 16 and 17
(Scheme 5) were prepared in three steps, starting from
2-amino-2-methyl-1,3-propanediol 12 which was pro-
tected as the tri¯uoroacetamide prior to dioxane forma-
tion, isomer separation and hydrolysis. The two amines
24 and 25 (Scheme 6) were made in ®ve steps from the 2-
azidomethyl-2-methyl-1,3-propanediol 19, the intermediate
amine 21 being tri¯uoroacetylated to facilitate separation
of the isomers which were in turn hydrolysed.
Schemes 7 and 8 give details of the six libraries prepared
from the acid and amine templates, respectively. Each
of the library compounds was prepared from 50 mg of
template with yields of between 10 and 100%.
Scheme 7. General method B for libraries 2 and 3. General method B:
(i) EDCI, HOBT, N,N-diisopropylamine, DMF.
Libraries 2 and 3 (Scheme 7) were prepared from acid
templates 10 and 11, respectively. For each template a
solution in DMF was dispensed into 32 ¯asks and coupled
with an appropriate amine using conventional amide
coupling reagents (EDCI/HOBT). Isolation and pur-
i®cation of the amide products was achieved by simple
aqueous work up with TLC to assess purity and MS to
characterise products (the amines utilised are shown in
Table 2).
clogP restriction of 5 were imposed on the compounds
in order to increase the probability for oral absorption.
Compounds in the library 1, which were prepared in a
similar manner to RPR132331 using commercially
available 1,3-diols (Table 1 shows the list of diols), is
depicted in Scheme 3. For this library (Scheme 3) a
solution of 6 in dichloromethane, was dispensed into 18
¯asks, treated with 2 equiv of diols 26 and allowed to
stand for 3 days during which time ecient dioxane
formation took place. Isolation and puri®cation was
carried out by preparative TLC with characterisation
largely by MS. In some cases two isomers (cis and trans
Libraries 4, 5, 6 and 7 (Scheme 8) were prepared from
templates 16, 17, 24 and 25, respectively. These were
dispensed, as THF solutions, into ¯asks and treated
with an appropriate electrophile leading to the generation
of amides, sulphonamides, thioureas and ureas. These
were isolated by preparative TLC and characterised by
MS (the electrophiles utilised are shown in Table 3).
This approach enabled the preparation of more than
200 compounds over a three months period, of which
190 passed the acceptance criteria (Table 4).
across the dioxane ring) were possible (when R1
6 Rˆ2).
Whenever possible, separation of these two isomers was
achieved using preparative TLC, with assignment of
structure according to NMR chemical shifts. This was
possible as ithas been shown previously 5 that the chemical
shiftof hydrogens on groups disposed axially atposition 5
of the dioxane ring are shifted 0.5 ppm down®eld rela-
tive to the corresponding equatorial isomer. The synth-
esis of the remaining six libraries required preparation
of two isomeric acid templates 10 and 11 (Scheme 4),
two isomeric amine templates 16 and 17 (Scheme 5) and
the two isomeric extended amines 24 and 25 (Scheme 6).
The two acids 10 and 11 (Scheme 4) were prepared in
two steps from the ester diol 8, following hydrolysis and
The syntheses of the two compounds RPR200765A
(identi®ed from library 3) and RPR201227A (identi®ed
from library 5), which were required for more detailed
evaluation, were investigated in some detail and are
shown in Scheme 9 and 10 respectively. Both com-
pounds were required as the trans isomers. The synthesis
Scheme 8. General methods C±G for libraries 4, 5, 6 and 7. General method C: (i) THF, rt. General method D: (ii) THF, re¯ux. General method E:
(iii) triethylamine, THF, rt. General method F: (iv) triethylamine, THF, re¯ux. General method G: (v) triethylamine, THF, re¯ux. General method
H: (vi) EDCI, HOBT, N,N-diisopropylamine, DMF.

542
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
of RPR200765A was more favourable than RPR201227A
as the required trans intermediate 11 was obtained as
the equivalent product (cis:transÐ1:1) whilstfor
RPR201227A the trans intermediate 17 was the minor
product( cis:transÐ3:1).
leastequivalentot SB203580. Samples prepared using
library techniques were used for all stages of the project
prior to drug disposition studies. In this way the inves-
tigation of the series was able to progress very rapidly
through the early stages of the biological assessment.
The number of compounds eliminated at early stages
was high, as can be seen from Table 4.
Biology
The 12 library samples showing in vivo activity <30 mg/kg
are shown in Table 5 with the corresponding in vitro, and
CYP1A1 induction data. These 12 compounds can be
seen to come from six of the seven libraries. However,
four of the 12 are the original amine templates for
libraries 4, 5, 6 and 7 which were included in the
respective library. Of the four compounds identi®ed
with an acceptable induction pro®le, two were templates
(16 [Lib4_1] and 17 [Lib5_1]) whilst the other two were
true library products: Lib3_28 and Lib5_14 (Table 5).
The two library compounds were resynthesised as
RPR200765A and RPR201227A, respectively (both as
mesylate salts). For each of the four, oral and intravenous
drug disposition studies were carried out in the rat.
Disappointingly the two templates 16 and 17 were not
found to produce signi®cant blood levels. This came as
a surprise as good in vivo activity had been seen, fol-
lowing oral dosing, in the mouse. There would appear
to be a species di€erence between rat and mouse in the
absorption of these primary amines. The project plan
utilised the rat as the species for both disease model
evaluation and toxicity and consequently 16 and 17
were abandoned. However, when converted to the
mesylate salt both RPR200765A and RPR201227A
showed excellentbioavailabiliyt wiht high blood levels.
The pharmacokinetic parameters for the two compounds
dosed orally at10 mg/kg or iv at1 mg/kg are compared
in Table 6.
The biological screening strategy is shown in Figure 2.
Potency cut-o€s were set to ensure compounds were at
Table 2. Amines (28, Scheme 7)
Listof amines
NH3
MeNH2
EtNH2
nPrNH2
cPrNH2
cPentNH2
cHexNH2
(nPr)2NH
(Me)2NH
HOCH2CH2NH2
HOCH2CH2CH2NH2
CH3OCH2CH2NH2
CH3OCH2CH2CH2NH2
2-PyCH2NH2
3-PyCH2NH2
4-PyCH2NH2
2-FurylCH2NH2
PhCH2CH2NH2
2-PyCH2CH2NH2
3-IndolylCH2CH2NH2
(Me)2NCH2CH2CH2NH2
(Me)2NCH2CH2NH2
Interestingly RPR201227A showed very high plasma con-
centrations resulting in a very low volume of distribution
which may re¯ect high plasma protein binding. Both com-
pounds were considered suitable for further evaluation.
Comparison of RPR201227A and RPR200765A
The two selected compounds were compared in vitro and
in vivo with each other. Inhibitory activity on various
kinases was ®rstperformed with results shown in Table 7.
Both compounds show p38 kinase inhibition at a level
similar to SB203580 (IC50 for SB203580=56 nM) and
both show good speci®city for p38 over other kinases.
The low micromolar potency against the src familly
kinase, lck has been noted previously with other potent
p38 inhibitors.15b
A full ED50 for mouse LPS-stimulated TNFa release
was performed for the two compounds and SB203580.
Results are presented in Figure 3 where it can be seen
that the compounds have potencies very similar to
SB203580.
RPR200765A and RPR201227A were both e€ective in the
SCW arthritis model in the Lewis rat using a prophylactic

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
Table 3. Electrophiles used for preparation of libraries 4, 5, 6 and 7 (Scheme 8)
543
Isocyanates
Thioisocyanates
Acid chlorides
Anhydrides
Sulphonyl chlorides
Acids
Drug safety
Table 4. Numbers of library compound accepted at early stages of
the project
Table 8 summarises the toxicity of the two compounds
RPR200765A and RPR201227A compared on Ames
test, micronucleus test, CYP 1A1 induction in vitro and
in vivo and in the guinea pig adrenotoxicity study.
Acceptance
criteria
Compounds
accepted
Library synthesis
Cellular assay
In vivo activity
CYP 1A1 induction
TLC single spot/M.S. correct
ED50<200 nM
ED50<30 mg/kg
190
24
12
4
Unlike SB203580, neither RPR201227A nor RPR200-
765A were able to induce chromosome damage or
induce CYP 1A1. RPR200765A was a very weak inhi-
bitor of ACAT activity and was also found to be free of
adrenotoxicity in the guinea pig at doses up to 800 mg/kg/
day. However, RPR201227A was a moderately active
inhibitor of ACAT and, more importantly, was found
to be highly toxic to guinea pigs (100 mg/kg/day)
requiring the study to be halted at an early stage. The
severity of the toxicity was such that it was decided to
abandon RPR201227A at this stage (see Table 8).
No induction
oral dosing regimen. Activities were again comparable
to SB203580 with all compounds showing ED50s on
paw swelling of 10±30 mg/kg/day (Fig. 4) and statisti-
cally signi®cantdose dependente€ecst on hisotlogical
parameters of 3±30 mg/kg/day (decrease in pannus area,
increase in area of intact joint surface and decrease
synovial hyperplasia and decrease in bone resorption
cavities, data not shown).
Scheme 9. Preparation of RPR200765A. (i) SOCl2, rt; (ii) morpholine, CH2Cl2; (iii) CH3SO3H, THF, rt.

544
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
Two further biological studies were carried out with
RPR200765A. Firstly, ecacy was evaluated in the
SCW arthritis model using a therapeutic oral dosing
regimen and dosing alongside SB203580 for comparative
purposes. This study showed that, as with prophylactic
dosing, RPR200765A and SB203580 produced signi®cant
e€ects on paw swelling and histological parameters at
10±30 mg/kg/day on both joint swelling and histological
parameters (Fig. 5). Finally, RPR200765A and SB203580
were examined for potential to inhibit CYP enzymes. As
can be seen from the results in Table 9, weak inhibition
of CYP 1A2 (IC50=4.2 mM) by RPR200765A was
observed but this was more than 80 times the IC50 seen
for p38 inhibition.
Scheme 10. Preparation of RPR201227A. (i) Benzoic acid, HATU,
DIPEA, DMF; (ii) CH3SO3H, THF, rt.
Figure 1. Structures of compounds relevant to this work.

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
545
Figure 2. Screening strategy adopted for optimisation of RPR132331. The stages are carried out sequentially and the acceptance criteria are quoted
ateach stage.
RPR200765AÐChemicaland physicalproperties
resynthesis and characterisation of compounds until
®rm candidates had been identi®ed. Considerable e€ort
was applied to the de®nition of the project objectives.
This enabled a rational screening strategy to be devised
and allowed key toxicity screens to be applied at a very
early stage (Fig. 2). The excellent oral bioavailability
and disease modifying activity of RPR200765A (both
prophylactically and therapeutically), the absence of
toxicity indicators (CYP 1A1 induction, mutagenicity
and guinea pig adrenotoxicity) combined with excellent
physical properties enabled RPR200765A to be proposed
and accepted as a development candidate for the
potential treatment of rheumatoid arthritis.
RPR200765A was highly stable in acidic, basic and
oxidative environments. At an early stage it had been
prepared as a mesylate salt to assist solubility and
bioavailability. The increase in solubility in distilled
water was great (free base 13 mg/L, mesylate 14 g/L).
This salt formed a stable, crystalline hydrate and only
one morphological form was found. The pharma-
ceutical properties of the mesylate salt were excellent.
A very ecientprocess rouet, was developed for bulk
preparation of the compound.26
Conclusions
Experimental
Chemistry
RPR132331 was identi®ed as a novel, weak inhibitor of
TNFa release from human monocytes. From this initial
lead, parallel synthesis and biological ®ltering techniques
were employed in the rapid identi®cation of a develop-
ment candidate: RPR200765A. The goal was to complete
the project within one year and towards this end a great
saving in chemical e€ortwas achieved by avoiding full
Analytical data were recorded for the compounds
described below using the following general procedures.
Proton NMR spectra were recorded on a Varian VXR
400; chemical shifts were recorded in ppm (d) from an
internal tetramethylsilane standard in deuterochloroform

546
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
Table 5. Library samples showing activity in the mouse LPS stimulated TNFa release assay
R
Library code
EC50 (nM)a
ED50 (mg/kg)b
10±30
CYP 1A1 inductionc
Inducer
SB203580
146 (Æ53)
Lib7_13
Lib7_1
Lib6_1
Lib4_1
Lib5_1
Lib1_12a
84 (Æ13)
38 (Æ8)
<30
10±30
10±33
10±30
<30
Inducer
Inducer
33 (Æ11)
55 (Æ20)
180 (Æ33)
200 (Æ80)
Inducer
Non-inducer
Non-inducer
Inducer
10±30
Lib5_14
Lib1_8b
110 (Æ24)
191 (Æ42)
10±30
10±30
Non-inducer
Inducer
Lib1_8a
Lib1_6
145 (Æ48)
117 (Æ13)
110 (Æ11)
<10
<30
<10
Inducer
Inducer
Lib3_28
Non-inducer
Lib1_5
115 (Æ21)
<30
Inducer
aRapid cellular screening was ®rst carried out using two concentrations only (100 and 300 nM, n=4). Full IC50 determinations were then carried out
for compounds showing more than 50% inhibition at 300 nM. Full protocol can be found in the Experimental.
bRapid screening was carried outin this assay using doses of 10 and 30 mg/kg only ( n=8). The criteria for acceptance was <30 mg/kg. SB203580
commonly produced results between 10 and 30 mg/kg with such a protocol.
cSee Experimental for protocols.
Table 6. Pharmacokinetic parameters for RPR200765A and RPR201227A in the rat
Compound
Oral dosing, 10 mg/kg
iv Dosing, 1 mg/kg
CpMax (ng.mLÀ1
)
AUC (h.ng.mLÀ1
)
Tmax (h)
T1/2 (h)
VD (L.kgÀ1
)
Cl (L.kgÀ1.hÀ1
)
T1/2 (h)
F (%)
RPR200765A
RPR201227A
5154
50134
11,111
480,463
0.25±0.5
1±2
2.8
6.2
0.7
0.1
1
0.013
1.6
5.6
50
64

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
547
Table 7. IC50s (mM) for di€erentkinases (see Experimental for assay
or deuterodimethyl sulphoxide as speci®ed below. Cou-
pling constants (J) were recorded in Hz. Mass spectra
(MS) were recorded on a VG 7070E/250 spectrometer
using chemical ionisation with NH3 as the carrier gas.
Microanalyses were performed on a Carlo-Erba 1106
microanalyser. Where analyses are indicated by the
symbols of the elements, results obtained were within
0.4% of the theoretical values. Melting points were
taken on an Electrothermal melting point apparatus
and are uncorrected. Reagents were purchased from
commercial sources. Anhydrous magnesium sulphate
was used routinely to dry the combined organic layers
from extractions. Solvent was routinely removed in
vacuo using a rotary evaporator.
conditions)a
Kinase
RPR200765A
RPR201227A
p38
0.050
>100
>40
>40
1.6
0.044
ꢀ100
>40
>40
0.4
ERK
ZAP70
SYK
Lck
aIC50 for SB203580=0.056 nM.
Commonly used abbreviations: THF (tetrahydrofuran),
DMF (dimethylformamide), DMSO (dimethyl sulph-
oxide), TLC (thin layer chromatography).
In many cases crude products were used, without further
puri®cation, as intermediates in subsequent reactions.
In such cases characterisation of the crude product was
carried through examination of the proton NMR and
identi®cation of an appropriate molecular ion in the
MS. For these syntheses the overall yields reported were
based on the original starting compound.
All library compounds were single spotby TLC and
characterised by MS and, in cases where there existed
the possibility of two isomers (cis and trans), the products
were prepared for screening as 10 mM solutions in deu-
terodimethyl sulphoxide and the relative orientation
1
assigned by H NMR prior to biological examination.
4-[2-Dimethoxymethyl-5-(4-¯uoro-phenyl)-1H-imidazol-
4-yl]-pyridine 6. (Scheme 1). A stirred solution of 5-
(4-¯uoro-phenyl)-4-(4-pyridyl)-imidazole25
(450 mg,
1.9 mmol, 1) in dry DMF (5 mL) was treated portion-
wise with sodium hydride (90 mg, 60% dispersion in
mineral oil). The mixture was stirred at room temperature
until the evolution of hydrogen stopped, then treated
dropwise with 2-(trimethylsilyl)ethoxymethyl chloride
(340 mg, 2 mmol). The mixture was then stirred at room
temperature for 1.5 h, the reaction mixture was treated
Figure 4. SCW arthritis model. E€ects of SB203580, RPR200765A
and RPR201227A on paw swelling following a prophylactic dosing
regimen (po, bid.). The paw swelling is measured twice daily and the
resultis given on the y-axis as mm per day. MeanÆS.E.M. *P<0.05,
**P<0.01, ANOVA with post hoc Dunnett's test compared to vehicle-
treated animals.
Figure 3. E€ectof SB203580, RPR200765A and RPR201227A on
LPS (0.1 mg/kg) induced TNF alpha release in male Balb/c mice.

548
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
Table 8. Summary of toxicological parameters determined
Compound
Ames
Micronucleus
CYP1A1
(in vitro)
CYP1A1
(in vivo)
ACAT
IC50 mM
Guinea pig
adrenotox.
SB203580
RPR200765A
RPR201227A
Negative up to 2500 mg/platea
Negative up to 2500 mg/platea
Negative up to 1000 mg/platea
Positive at 50 mg/mL and above
Negative up to 25 g/mLa
Negative up to 75 mg/mLa
+
À
À
+
À
À
40
62
2.5
ND
À
b
aLowest insoluble concentration.
bStudy abandoned due to animal death.
Figure 5. E€ects of RPR200765A administered in a therapeutic regimen on histological parameters. RPR200765A was dosed at 3, 10, 30 and
100 mg/kg/day (po, bid.), SB203580 used as a comparison at 30 mg/kg/day (po, bid.) and the standard dexamethasone (Dex) at 0.5 mg/kg/day. The
histology was assessed in joints obtained at the study termination (day 28) and quanti®ed in terms of joint area (panel A), pannus (panel B), synovial
hyperplasia (panel C) and resorption index (panel D) by histomorphometric analysis. Results represent means Æ S.E.M. *P<0.05, **P<0.01,
***P<0.001 compared to relevant vehicle control.
with water (0.2 mL) and evaporated. The residual oil
was then partitioned between ethyl acetate (5 mL) and
water (5 mL) and the aqueous phase was extracted twice
further with ethyl acetate. The combined organic phases
were washed with brine, dried and evaporated. The
residual oil was subjected to ¯ash chromatography on
silica gel, eluting with a mixture of methanol:dichloro-
methane (98:2, v/v), to give a mixture of the silyl
derivatives 4-[5-(4-¯uoro-phenyl)-[1 or 3]-[(2-(trimethyl-
silyl)ethoxy)methyl]-1H-imidazol-4-yl]-pyridine (0.529 g,
1.4 mmol, 2 and 3) as a yellow oil. This intermediate was
dissolved in dry THF (5 mL) and treated with a solution
of n-butyllithium in hexane (0.7 mL, 2.1 M) to give a
dark green solution which was stirred at À78 ꢁC for
15 min before treating with a solution of N-formyl-
morpholine (160 mg, 1.4 mmol) in THF (10 mL). After
stirring for 15 min at À78 ꢁC, the mixture was allowed
to warm to room temperature and stirred for a further
1 h. The reaction mixture was quenched with water
(10 mL) and extracted with ethyl acetate (3Â30 mL).
The combined extracts were dried and evaporated. The
residual oil was subjected to ¯ash chromatography on
silica gel, eluting with ethyl acetate, to give a mixture of
Table 9. IC50 (mM) for inhibition of various human CYP enzymes
Compound
CYP 3A4
CYP 2D6
CYP 1A2
CYP 2C9
RPR200765
SB203580
20
4.4
>50
28
4.2
2.6
12
0.44

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
549
formylated products 4-[5-(4-¯uoro-phenyl)-2-formyl-[1
or 3]-[(2-(trimethylsilyl)ethoxy)methyl]-1H-imidazol-4-yl]-
pyridine (525 mg, 1.3 mmol, 4 and 5) as a yellow oil.
This intermediate was dissolved in methanol (10 mL)
and treated with trimethylorthoformate (5 mL) then
with 4-toluenesulphonic acid (0.39 g). The reaction mixture
was re¯uxed for 5 h, cooled to room temperature and
evaporated under reduced pressure. The residue was
partitioned between ethyl acetate (30 mL) and saturated
aqueous sodium bicarbonate solution (30 mL). The aqu-
eous phase was extracted four times with ethyl acetate
(20 mL). The combined organic phases were washed
with water (15 mL), brine (15 mL), dried over magnesium
sulphate and evaporated. The residual oil was subjected
to ¯ash chromatography on silica gel eluting with a
mixture of dichloromethane:methanol (8:1, v/v) to give
6 (0.344 g, 58% overall) as a white solid having mp 186±
189 ꢁC. 1H NMR (DMSO-d6) 8.35 (d, J=6 Hz, 2H),
7.43 (d, J=6 Hz, 2H), 7.36 (dd, J=8 Hz and J=4 Hz,
2H), 7.19 (t, J=8 Hz, 2H), 5.39 (s, 1H), 3.34 (s, 6H).
Anal. (C17H16FN3O2) C, H, N.
2.4 mmol, 8) and 4-toluenesulphonic acid (520 mg,
3 mmol) in dry THF (20 mL) was heated at re¯ux for
6 h. The mixture was allowed to cool to room tempera-
ture then partitioned between ethyl acetate (20 mL) and
saturated sodium bicarbonate solution (20 mL). The
organic phase was washed twice with water (2Â10 mL),
brine (10 mL), dried and evaporated. The residue was
subjected to chromatography on silica gel eluting with a
mixture of dichloromethane:methanol (9:1, v/v) to
provide c/t-2-[5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imid-
azol-2-yl]-5-methyl-[1,3]dioxane-r-5-carboxylic acid methyl
esters (cis- and trans-isomers) as a cream solid (340 mg,
0.86 mmol, 9). This mixture of intermediate esters was
dissolved in methanol (15 mL), treated with aqueous
sodium hydroxide solution (1.71 mL, 1 N) and heated at
re¯ux for 7 h. The reaction mixture was cooled and
evaporated. The residual cream powder was dissolved in
methanol (10 mL) and the solution acidi®ed to pH 5±6
by addition of glacial acetic acid. The resulting white
precipitate was ®ltered and washed with pentane to give
the trans isomer 11 (0.11 g, 24% overall) as a white solid
having mp 265±267 ꢁC (d). H NMR (DMSO-d6) 8.50
1
4-[2-(5,5-Dimethyl-[1,3]dioxan-2-yl)-5-(4-¯uoro-phenyl)-
1H-imidazol-4-yl]-pyridine RPR132331. (Scheme 2). A
stirred solution of 4-[2-dimethoxymethyl-5-(4-¯uoro-
phenyl)-1H-imidazol-4(5)-yl]-pyridine (5.0 g, 15.9 mmol,
6) and 2,2-dimethyl-1,3-propanediol (3.33 g, 31.8 mmol,
7) and 4-toluenesulphonic acid (6.8 g, 39.7 mmol) in dry
THF (200 mL) was heated at re¯ux for 6 h. The mixture
was allowed to cool to room temperature then parti-
tioned between ethyl acetate (200 mL) and saturated
sodium bicarbonate solution (200 mL). The organic
phase was washed with water (100 mL), brine (100 mL),
then dried and evaporated to give RPR132331 (3.36 g,
(m, 2H), 7.53±7.28 (m, 6H), 5.51 (s, 1H), 4.08 (d,
J=10 Hz, 2H), 3.90 (d, J=10 Hz, 2H), 1.48 (s, 3H,
CH3-ax). Anal. (C20H18FN3O4) C, H, N. The ®ltrate
plus washings were separated by ¯ash chromatography
on silica gel eluting with a mixture of dichloromethane:
methanol (9:1, v/v) to give the cis isomer 10 (0.14 g,
ꢁ
1
30% overall) as a white solid having mp 290 C (d). H
NMR (DMSO-d6) 8.44 (m, 2H), 7.46 (dd, J=8 Hz and
J=4 Hz, 2H), 7.36 (d, J=8 Hz, 2H), 7.30±7.21 (m, 2H),
5.66 (s, 1H), 4.47 (d, J=12 Hz, 2H), 3.71 (d, J=12 Hz,
ꢂ
2H), 0.94 (s, 3H, CH3-eq). Anal. (C20H18FN3O4
CH3OH) C, H, N.
61%) as a cream solid having mp 248±249 ꢁC (d). H
1
NMR (DMSO-d6) 8.72 (d, J=8 Hz, 2H), 7.95 (d,
J=8 Hz, 2H), 7.62 (dd, J=8 Hz and J=4 Hz, 2H), 7.40
(t, J=8 Hz, 2H), 5.68 (s, 1H), 3.72 (s, 4H), 1.25 (s, 3H,
CH3-ax), 0.79 (s, 3H, CH3-eq). Anal. (C20H20FN3-
c-5-Methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imid-
azol-2-yl}-[1,3]dioxan-r-5-ylamine cis-isomer 16 and t-5-
methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-
2-yl}-[1,3]dioxan-r-5-ylamine trans-isomer 17. (Scheme
5). A stirred solution of 2-amino-2-methyl-1,3-propane-
diol (7.5 g, 71.4 mmol, 12) in dry DMF (150 mL), at
room temperature, was treated with potassium carbonate
(11.2 g). After stirring at room temperature for 15 min
the mixture was treated dropwise with tri¯uoroacetic
anhydride (11.5 mL) and stirred for a further 18 h. The
reaction mixture was evaporated and the residue was
subjected to ¯ash chromatography on silica gel eluting
with a mixture of dichloromethane:methanol (95:5,
v/v) to give 2-methyl-2-tri¯uoroacetamido-1,3-propane-
diol (7.38 g, 36.7 mmol, 13). This intermediate was dis-
solved in dry THF (200 mL) and treated with 4-[2-
dimethoxymethyl-5-(4-¯uoro-phenyl)-1H-imidazol-4-yl]-
pyridine (5.75 g, 18.3 mmol, 6) and 4-toluenesulphonic
acid (8.03 g, 46.7 mmol) and the mixture heated at re¯ux
temperature for 6 h. The mixture was cooled, treated
with saturated sodium bicarbonate solution (200 mL),
and extracted with ethyl acetate (200 mL). The organic
phase was washed with water (2Â100 mL), brine (100 mL),
dried and evaporated. The residue was subjected to
chromatography on silica gel eluting with a mixture of
dichloromethane:methanol (95:5, v/v) to give c-2,2,
2-tri¯uoro-N-[5-methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-
yl-1H-imidazol-2-yl}-[1,3]dioxan-r-5-yl]-acetamide 14
ꢂ
O2 0.5H2O) C, H, N.
Generalmethod A. Preparation of compounds in ilbrary 1.
(Scheme 3). A solution of 4-[2-dimethoxymethyl-5-(4-
¯uoro-phenyl)-1H-imidazol-4-yl]-pyridine
6
(50 mg,
0.16 mmol) and 4-toluenesulphonic acid (70 mg,
0.4 mmol) in dichloromethane (5 mL) was treated with
an appropriately substituted 1,3-propanediol (2 equiv,
26, Table 1) and allowed to stand for 3 days. Each
reaction mixture was then washed with saturated
sodium bicarbonate solution (5 mL), dried and evapo-
rated. The crude residues were then subjected to pre-
parative thick layer chromatography on silica gel
eluting with mixtures of dichloromethane/methanol to
provide the library product (27) which was prepared, for
biological evaluation, as a 10mM solution in DMSO.
c-2-[5-(4-Fluoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl]-5-
methyl-[1,3]dioxane-r-5-carboxylic acid 10 and t-2-[5-(4-
¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl]-5-methyl-
[1,3]dioxane-r-5-carboxylic acid 11. (Scheme 4). A stirred
solution of 4-[2-dimethoxymethyl-5-(4-¯uoro-phenyl)-
1H-imidazol-4(5)-yl]-pyridine (370 mg, 1.2 mmol, 6),
methyl 2,2-bis(hydroxymethyl)propionate (380 mg,

550
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
(cis-isomer, 4.3 g, 9.5 mmol) and t-2,2,2-tri¯uoro-N-[5-
methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imida-
zol-2-yl}-[1,3]di-oxan-r-5-yl]-acetamide 15 (trans-isomer,
1.62g, 3.6 mmol) both as light brown gums. Both inter-
mediates were dissolved separately in methanol (cis iso-
mer: 300 mL; trans isomer: 100 mL) and treated with
potassium carbonate (cis isomer: 2.6 g, 19 mmol; trans
isomer: 1 g, 7.2 mmol) and water (cis isomer: 7.5 mL; trans
isomer: 2.5 mL). The mixtures were heated for 8 h, eva-
porated, and residual water removed by azeotroping with
toluene. Each residue was then subjected to ¯ash chro-
matography on silica gel eluting with a mixture of
dichloromethane:methanol (4:1,v/v) to provide the two
title products as white solids: the cis-product 16
isomers was suspended in dichloromethane (50 mL) and
treated with triethylamine (1 mL) and tri¯uoroacetic
anhydride (1 mL). After stirring at room temperature
for 4.5 h the reaction mixture was partitioned between
ethyl acetate (50 mL) and water (50 mL). The organic
phase was washed with water (50 mL), brine (50 mL),
dried and evaporated. The residue was subjected to
preparative HPLC, using methanol:water 40:60 v/v to
5:95 v/v over 20 min to give c-2,2,2-tri¯uoro-N-[5-
methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imid-
azol-2-yl}-[1,3]dioxan-r-5-ylmethyl]-acetamide, cis-isomer
22 (750 mg, 1.6 mmol, RT=12 min) and t-2,2,2-tri¯uoro-
N-[5-methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-
imidazol-2-yl}-[1,3]dioxan-r-5-ylmethyl]-acetamide, trans-
isomer 23 (300 mg, 0.64 mmol, RT=11 min). Both
intermediates were dissolved separately in methanol (cis
isomer: 100 mL; trans isomer: 50 mL) and treated with
potassium carbonate (cis isomer: 1.11 g, 8 mmol; trans
isomer: 440 mg, 3.2 mmol) and water (cis isomer:
2.5 mL; trans isomer: 1.2 mL). The mixtures were heated
for 8 h, evaporated and residual water removed by
azeotroping with toluene. Each residue was subjected
to ¯ash chromatography on silica gel eluting with a mix-
ture of dichloromethane:pentane:methanol:concentrated
ammonia (55:25:18:2, v/v/v/v) to provide the two title
products as white solids: the cis-product 24 (370 mg,
23% overall) having mp 203±205 ꢁC. 1H NMR (DMSO-
d6) 8.45 (d, J=6 Hz, 2H), 7.46 (dd, J=8 Hz and
J=4 Hz, 2H), 7.36 (d, J=6 Hz, 2H), 7.28 (t, J=8 Hz,
2H), 5.58 (s, 1H), 3.93 (d, J=12 Hz, 2H), 3.60 (d,
J=12 Hz, 2H), 2.88 (s, 2H), 0.65 (s, 3H, CH3-eq). Anal.
(C20H18FN3O4) C, H, N and the trans-product 25
(150 mg, 9% overall) having mp 158Ð165 ꢁC. 1H NMR
(DMSO-d6) 8.44 (d, J=6 Hz, 2H), 7.48 (dd, J=8 Hz
and J=4 Hz, 2H), 7.37 (d, J=6 Hz, 2H), 7.27 (t,
J=8 Hz, 2H), 5.53 (s, 1H), 3.77 (d, J=12 Hz, 2H), 3.72
(d, J=12 Hz, 2H), 2.65 (s, 2H), 1.21 (s, 3H, CH3-ax).
Anal. (C20H18FN3O4) C, H, N.
(2.4 g, 37.7% overall) having mp 194 ꢁC (d). H NMR
1
(DMSO-d6) 8.47 (m, 2H), 7.51 (dd, J=8 Hz and
J=4 Hz, 2H), 7.39 (d, J=6 Hz, 2H), 7.31 (m, 2H), 5.64
(s, 1H), 3.83 (d, J=12 Hz, 2H), 3.68 (d, J=12 Hz, 2H),
ꢂ
0.90 (s, 3H, CH3-eq). Anal. (C20H18FN3O4 0.25H2O) C,
H, N and the trans-product 17 (0.93 g, 14.2% overall)
having mp 254 ꢁC (d). H NMR (DMSO-d6) 8.36 (m,
1
2H), 7.43 (m, 2H), 7.32 (d, J=6 Hz, 2H), 7.36 (m, 2H),
5.47 (s, 1H), 3.63 (d, J=10 Hz, 2H), 3.54 (d, J=10 Hz,
ꢂ
2H), 1.26 (s, 3H, CH3-ax). Anal. (C20H18FN3O4
0.25H2O) C, H, N.
c-C-[5-Methyl-2-(5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-
imidazol-2-yl)-[1,3]dioxan-r-5-yl]-methylamine cis-isomer 24
and t-C-[5-methyl-2-(5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-
imidazol-2-yl)-[1,3]dioxan-r-5-yl]-methylamine trans-isomer
25. (Scheme 6). 5-Azidomethyl-2-dimethyl-5-methyl-1,3-
dioxane27 (1.48 g, 8 mmol, 18) was dissolved in dry THF
(10 mL), treated with concentrated hydrochloric acid
(0.3 mL) and water (0.8 mL) before heating at re¯ux
temperature for 1 h. The mixture was then cooled and
evaporated to give 2-azidomethyl-2-methyl-1,3-propa-
nediol (1.3 g, 8.9 mmol, 19) as a colourless oil. This
intermediate was dissolved in dry THF (50 mL) and
treated with 4-[2-dimethoxymethyl-5-(4-¯uoro-phenyl)-
1H-imidazol-4-yl]-pyridine (1.4 g, 4.4 mmol, 6) and 4-
toluenesulphonic acid (1.9 g, 11.3 mmol) and the mixture
heated at re¯ux temperature for 6 h. The mixture was
cooled, treated with saturated sodium bicarbonate
solution (50 mL) and extracted with ethyl acetate
(50 mL). The organic phase was washed with water
(2Â25 mL), brine (25 mL), dried and evaporated. The
residue was subjected to ¯ash chromatography on
silica gel eluting with a mixture of dichloromethane:
methanol (9:1 v/v) to give 4-[2-(c/t-5-azidomethyl-5-
methyl-[1,3]dioxan-r-2-yl)-5-(4-¯uoro-phenyl)-1H-imid-
azol-4-yl]-pyridine, cis- and trans-isomers 20 (1.56 g,
3.9 mmol) as a white solid. This intermediate mixture of
isomers was dissolved in methanol (100 mL), treated
with ammonium formate (1 g) and 10% palladium
on activated carbon (0.15 g). The mixture was stirred
for 3.5 h, ®ltered through diatomaceous earth and eva-
porated. The residual orange solid was subjected to
¯ash chromatography on silica gel eluting with a mix-
ture of dichloromethane:pentane:methanol:concentrated
ammonia (55:25:18:2 v/v/v/v) to give a mixture of C-[5-
methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-
yl}-[1,3]dioxan-5-yl]-methylamine, cis- and trans-isomers
21 (0.980 g, 2.7 mmol) as a yellow solid. This mixture of
Generalmethod B. Preparation of compounds in libraries 2
and 3. (Scheme 7). A solution of cis or trans-2-[5-(4-
¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl]-5-methyl-
[1,3]dioxane-5-carboxylic acid (cis- or trans-isomer) 10
or 11 (50 mg, 0.13 mmol), an appropriate primary or
secondary amine (1.1 equiv, 28 Table 2), 1-(3-dimethyl-
amino-propyl)-3-ethylcarbodiimide (1.1 equiv), 1-
hydroxybenzotriazol hydrate (1.1 equiv) and N,N-
diisopropylethylamine (3 equiv) in dry DMF (5 mL)
was stirred at room temperature for 18 h. For each
reaction the mixture was evaporated and the residue
partitioned between ethyl acetate and water. The
organic phase was washed with brine and evaporated to
give the required library compound 29 or 30 which was
prepared, for biological evaluation, as a 10 mM solution
in DMSO.
Generalmethod C. Preparation of ureas in ilbraries 4, 5,
6 and 7. (Scheme 8). A solution of 5-methyl-2-{5-(4-
¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl}-[1,3]di-
oxan-5-ylamine (cis- or trans-isomer) 16 or 17 (50 mg,
0.13 mmol) or C-[5-methyl-2-(5-(4-¯uoro-phenyl)-4-pyr-
idin-4-yl-1H-imidazol-2-yl)-[1,3]dioxan-5-yl]-methylamine
(cis- or trans-isomer) 24 or 25 (50 mg, 0.13 mmol) and

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
551
an appropriately substituted isocyanate (1 equiv, Table
3) in dry THF was stirred at room temperature for
30 min. For each reaction the mixture was evaporated
and the residue subjected to preparative thick layer
chromatography on silica gel, eluting with a mixture of
dichloromethane:methanol (9:1 v/v), and the required
library compound 31 prepared, for biological evalua-
tion, as a 10 mM solution in DMSO.
[1,3]dioxan-5-ylamine (cis- or trans-isomer) 16 or 17
(50 mg, 0.13mmol) or C-[5-methyl-2-(5-(4-¯uoro-phenyl)-
4-pyridin-4-yl-1H-imidazol-2-yl)-[1,3]dioxan-5-yl]-methyl-
amine (cis- or trans-isomer) 24 or 25 (50 mg, 0.13 mmol)
and triethylamine (1 equiv) in dry THF (5 mL) was
treated with an appropriately substituted sulphonyl
chloride (1 equiv, Table 3) and the mixture heated at
re¯ux temperature for 8 h. The reaction mixture was
evaporated and the residue subjected to preparative
thick layer chromatography on silica gel, eluting with a
mixture of dichloromethane:methanol (9:1 v/v). The
required library compound 35 was isolated and pre-
pared, for biological evaluation, as a 10 mM solution in
dimethylsulphoxide.
Generalmethod D. Preparation of thioureas in ilbraries
4, 5, 6 and 7. (Scheme 8). A solution of 5-methyl-2-{5-
(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl}-[1,3]di-
oxan-5-ylamine (cis- or trans-isomer) 16 or 17 (50 mg,
0.13 mmol) or C-[5-methyl-2-(5-(4-¯uoro-phenyl)-4-pyr-
idin-4-yl-1H-imidazol-2-yl)-[1,3]dioxan-5-yl]-methylamine
24 or 25 (cis- or trans-isomer) (50 mg, 0.13 mmol) and
an appropriately substituted isothiocyanate (1 equiv,
Table 3) in dry THF (5 mL) was heated at re¯ux for
18 h. For each reaction the mixture was evaporated and
the residue subjected to preparative thick layer chromato-
graphy on silica gel, eluting with a mixture of dichloro-
methane:methanol (9:1 v/v), and the required library
compound 32 isolated and prepared, for biological eva-
luation, as a 10 mM solution in DMSO.
Generalmethod H. Preparation of basic amides in ilbr-
aries 4, 5, 6 and 7. (Scheme 8). A solution of 5-methyl-
2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl}-
[1,3]dioxan-5-ylamine (cis- or trans-isomer) 16 or 17
(50 mg, 0.13mmol) or C-[5-methyl-2-(5-(4-¯uoro-phenyl)-
4-pyridin-4-yl-1H-imidazol-2-yl)-[1,3]dioxan-5-yl]-methyl-
amine (cis- or trans-isomer) 24 or 25 (50 mg, 0.13 mmol)
an appropriate carboxylic acid (1.1 equiv, Table 3), 1-(3-
dimethylamino-propyl)-3-ethylcarbodiimide (1.1 equiv-
alents), 1-hydroxybenzotriazol hydrate (1.1 equiv) and
N,N-diisopropylethylamine (3 equiv) in dry DMF (5 mL)
was stirred at room temperature for 18 h. For each
reaction the mixture was evaporated and the residue
partitioned between ethyl acetate and sodium bicarbo-
nate solution. The organic phase was dried, evaporated
and the residue subjected to preparative thick layer
chromatography on silica gel, eluting with dichloro-
methane:methanol, to provide the required library
compound 36 which was prepared, for biological eva-
luation, as a 10 mM solution in DMSO.
Generalmethod E. Preparation of neutralamides in ilbr-
aries 4, 5, 6 and 7. (Scheme 8). A solution of 5-methyl-
2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl}-
[1,3]dioxan-5-ylamine (cis- or trans-isomer) 16 or 17
(50 mg, 0.13mmol) or C-[5-methyl-2-(5-(4-¯uoro-phenyl)-
4-pyridin-4-yl-1H-imidazol-2-yl)-[1,3]dioxan-5-yl]-methyl-
amine 24 or 25 (cis- or trans-isomer) (50 mg, 0.13 mmol)
and triethylamine (1 equiv) in dry THF (5 mL) was
treated with an appropriately substituted acid chloride
(1 equiv, Table 3) and the mixture stirred for 18 h at
room temperature. For each reaction the mixture was
evaporated and the residue subjected to preparative
thick layer chromatography on silica gel, eluting with a
mixture of dichloromethane:methanol (9:1 v/v), and the
required library compound 33 isolated and prepared, for
biological evaluation, as a 10 mM solution in DMSO.
In some cases the reaction was performed with an
amino acid protected as a benzyloxycarbonyl derivative.
The products from these reactions were subsequently
treated with palladium, 5% activated on carbon and
stirred at room temperature under a hydrogen atmo-
sphere for 8 h, ®ltering through a pad of diatomaceous
earth, evaporated and the residue subjected to preparative
thick layer chromatography on silica gel, eluting with
dichloromethane:methanol (7:3 v/v) to provide the
required primary amine productwhich was prepared,
for biological evaluation, as a 10 mM solution in
DMSO.
Generalmethod F. Preparation of acidic amides in il-
braries 4, 5, 6 and 7. (Scheme 8). A solution of 5-
methyl-2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2
-yl}- [1,3]dioxan-5-ylamine (cis- or trans-isomer) 16 or 17
(50 mg, 0.13mmol) or C-[5-methyl-2-(5-(4-¯uoro-phenyl)-4-
pyridin-4-yl-1H-imidazol-2-yl)-[1,3]dioxan-5-yl]-methyl-
amine (cis- or trans-isomer) 24 or 25 (50 mg, 0.13 mmol)
and triethylamine (1.2 equiv) in dry THF (5 mL) was
treated with an appropriately substituted cyclic acid
anhydride (1 equiv, Table 3) and the mixture heated at
re¯ux temperature for 8 h. For each reaction the mix-
ture was evaporated and the residue subjected to pre-
parative thick layer chromatography on silica gel,
eluting with a mixture of dichloromethane:methanol (9:1
v/v), and the required library compound 34 isolated and
prepared, for biological evaluation, as a 10 mM solution
in DMSO.
{t-2-[4-(4-Fluoro-phenyl)-5-pyridin-4-yl-1H-imidazol-2-yl]-
5-methyl-[1,3]dioxan-r-5-yl}-morpholin-4-yl-methanone
methane sulphonic acid salt, trans-isomer, RPR200765A.
(Scheme 9). t-2-[5-(4-Fluoro-phenyl)-4-pyridin-4-yl-1H-
imidazol-2-yl]-r-5-methyl-[1,3]dioxane-5-carboxylic acid,
trans-isomer 11 (7.70 g, 20 mmol) was added portion-
wise to stirred thionyl chloride (50 mL). The reaction
mixture was stirred at room temperature for 1.25 h,
evaporated and the residue azeotroped with dry toluene
to yield the crude acid chloride 38 which was dissolved in
dry dichloromethane (150 mL) and treated with morpho-
line (30 mL). The mixture was stirred at room temperature
for 2.5 h, evaporated and the residue partitioned between
ethyl acetate (250 mL) and saturated sodium bicarbonate
General method G. Preparation of sulphonamides in libr-
aries 4, 5, 6 and 7. (Scheme 8). A solution of 5-methyl-
2-{5-(4-¯uoro-phenyl)-4-pyridin-4-yl-1H-imidazol-2-yl}-

552
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
(200 mL). The insoluble product at the interface was
®ltered o€ and washed with water (20 mL), methanol
(10 mL), diethyl ether (20 mL) to give the free base of
the title compound (7.43 g, 16.9 mmol) which was dis-
solved in hot THF (500 mL) and treated with a solution
of methane sulphonic acid (1.578 g) in THF. After
standing at room temperature for 18 h the reaction
mixture was ®ltered and the solid recrystallised from
acetonitrile with hot ®ltration through Celite to give
RPR200765A (6.00 g, 53% overall) as a pale yellow
(sheep anti-rabbit IgG) with addition of the DELFIA
enhancementsoluiton resulitng in ¯uorescence. ERK
was measured using a [33P] ATP ®ltration assay format
using for substrates myelin basic protein. ZAP-70, Syk
and Lck kinase activities were measured using the
homogeneous time-resolved ¯uorescence methodology
(HTRF) with the catalytic domains of each of the tyrosine
kinases, biotinylated, speci®c peptide substrates, strep-
tavidin-linked APC and europium cryptate-conjugated
anti-phosphotyrosine antibody. Results represent means
Æ S.E.M. (n=3).
crystalline solid having mp 242±246 ꢁC (d). H NMR
1
(DMSO-d6) 8.65 (d, J=8 Hz, 2H), 7.86 (d, J=8 Hz,
2H), 7.60 (dd, J=8 Hz and J=4 Hz, 2H), 7.40 (t,
J=8 Hz, 2H), 5.69 (s, 1H), 4.12 (s, 4H), 3.59±3.50 (m,
8H), 2.31 (s, 3H, CH3SO3), 1.58 (s, 3H, CH3-ax). Anal.
Monocyte TNFꢀ release assay. Adherenthuman
monocytes (100,000 cells/well) were incubated with LPS
(10 ng/mL) in the absence and presence of compound
for 18 h. Individual experiments were carried out in
quadruplicate samples. TNFa was measured by sandwich
ELISA and IC50 values calculated for the activity of
individual compounds. IC50 values shown from repeat
experiments are meansÆS.E.M. (n=3). means Æ S.E.M.
(n=3).
ꢂ
(C24H25FN4O4 CH3SO3H) C, H, N.
t-N-{2-[4-(4-Fluoro-phenyl)-5-pyridin-4-yl-1H-imidazol-2-
yl]-5-methyl-(1,3)dioxan-r-5-yl}-benzamide, methane sulph-
onic acid salt, trans-isomer RPR201227A. (Scheme 10).
A stirred suspension of 5-methyl-2-{5-(4-¯uoro-phenyl)-
4-pyridin-4-yl-1H-imidazol-2-yl}-[1,3]dioxan-5-ylamine,
trans isomer 17 (1.76 g, 5 mmol), benzoic acid (0.67 g,
5.5 mmol) and diisopropylethylamine (1.39 mL) in dry
DMF (50 mL) was treated with {0-(7-azabenzotriazol-1-
yl)-1,1,3,3-tetramethyluronium hexa¯uorophosphate}
(1.88 g, HATU) under a nitrogen atmosphere. The mixture
was stirred at room temperature for 2 h and evaporated
to dryness. The residue was partitioned between ethyl
acetate (70 mL) and saturated sodium bicarbonate (50 mL).
The organic phase was washed twice with water (50 mL),
then with brine (30mL) and then evaporated. The residue
was subjected to ¯ash chromatography on silica gel
eluting with ethyl acetate to give the free base of the title
product(0.85 g, 1.85 mmol). This was dissolved in THF
(100 mL) and treated with methane sulphonic acid
(0.178 g, 1.85 mmol). The reaction mixture was agitated
for 5 min then evaporated to dryness and dried under
high vacuum. The residual solid was recrystallised from
ethyl acetate containing a minimum volume of acetonitrile
to give RPR201227A (900 mg, 32% overall) as a yellow
crystalline solid having mp 165±169 ꢁC. 1H NMR
(DMSO-d6) 8.65 (d, J=8 Hz, 2H), 8.03 (s, 1H), 7.87±
7.80 (m, 4H), 7.63±7.51 (m, 3H), 7.47 (t, J=8 Hz, 2H),
7.39 (t, J=8 Hz, 2H), 5.77 (s, 1H), 4.31 (d, J=10 Hz,
2H), 4.12 (d, J=10 Hz, 2H), 2.31 (s, 3H, CH3SO3), 1.71
Mouse TNFꢀ release assay. Compound was adminis-
tered orally to balb/c mice 30 min prior to LPS (0.1 mg/
kg ip) challenge. Serum TNFa levels were determined
90 min after LPS insult. Results represent means Æ
S.E.M. (n=3).
Hepatocyte CYP induction assay. Primary hepatocytes
were isolated from Sprague±Dawley rats, cultured in
6-well plates and treated with vehicle (0.25% DMSO)
or compound (30, 50 and 100 mM) for a period of 3
days. At the end of the incubation, cells were harvested,
homogenised and the post-nuclear fractions isolated.
Content of CYP isozymes (1A1, 1A2, 2B1 and 2B2)
were evaluated by Western blot using isozyme speci®c
antibodies.
Rat drug distribution studies. Three rats were dosed
orally (10 mg/kg) with a suspension of compound in
methylcellulose or iv (1 mg/kg) with a solution of com-
pound in N-methylpyrrolidone. Blood samples were
taken at intervals over 24 h and analysed for compound.
Averaged results are reported with Æ S.E.M. (n=3).
Rat CYP induction assay. Compound was dosed orally
to three male rats at 100 and 200 mg/kg/day for 5 days.
Animals were sacri®ced and liver CYP (1A1, 1A2, 2B1,
2B2, 3A1 and 2E1) levels assessed by Western blot analysis.
ꢂ
(s, 3H, CH3-ax). Anal. (C26H23FN4O3 CH3SO3H) C,
H, N.
Rat streptococcal cell wall (SCW) induced arthritis
model. This model was performed using Lewis Rats in
either a prophylactic or therapeutic regimen. On day 0
an intra-articular injection of 10 mg of SCW 100P frac-
tion (in 10 mL) was made into the tibiotarsal joint of one
hind limb of anaesthetised female rat. Any animals
which had notdeveloped a swollen ankle 2 days laetr
were excluded from the study. Challenge with iv SCW
was made on day 21 and compounds given on day 20±
24 (po, bid.) for the prophylactic studies and 21±24 for
the therapeutic studies. Paw swelling was calculated by
summation of daily measurement of the paw medial and
lateral malleoli width post challenge with iv SCW until
Biological Methods
Kinases assay
The p38 enzyme assay is carried outatroom temperature
for 1 h, using 40ng/well of the mouse enzyme. The sub-
strate, 50mg/mL ATF-2 is coated onto 96-well plates, the
assay is carried outin 25 mM Hepes bu€er, pH 7.7
containing 25 mM magnesium chloride, 2 mM dithio-
threitol, 1 mM sodium orthovanadate and 100 mM ATP.
Phosphorylated ATF-2 is quantitated using a phospho-
speci®c ATF-2 primary antibody (rabbit anti-human)
followed by a europium-labelled secondary antibody

I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
553
termination of study. Radiography of tibio-tarsal joints
Acknowledgements
was undertaken using a Phillips Practix X-ray unit.
Histopathology was performed on formalin-®xed tissues,
processed and stained with Alcian Blue. Histomorpho-
metry of histological structures was performed by image
analysis using an Apple Quadra 700 computer attached
to an Olympus BX50 microscope using the Image-
GrabberTM program and analysed by OptilabTM soft-
ware (both ME Electronics Ltd, Reading, UK).
Structures examined were: una€ected joint area (area of
histologically normal tibio-tarsal tissues); synovial
hyperplasia (the intrusion of activated synovial villi into
the articular space); pannus (total pannus area within
the sub-chondral bone); bone resorption index (the
number of activated cavities divided by the total cavity
number). Compound e€ects on paw swelling indices
were compared to vehicle groups using ANOVA and
Dunnetts test (n=3). Radiographic and histological
indices were analysed using the Mann±Whitney U test.
Groups of 10 rats were used for each dosing protocol.
Mean Æ S.E.M. * P<0.05, ** P<0.01, ANOVA with post
hoc Dunnett's test compared to vehicle-treated animals.
None of this work would have been possible without the
skill and dedication of the following synthetic chemists:
Adnan Al-Shaar, Mustafa Bhimani, Imtiaz Ahmed,
Andrew Bootle-Wilbraham, and Andrew Emm.
References and Notes
1. Figures for the prevalence of rheumatoid arthritis were
obtained from http://www.MedicineNet.com.
2. Lee, J. C.; Badger, A. M.; Griswold, D. E.; Dunnington,
D.; Truneh, A.; Votta, B.; White, J. R.; Young, P. R.; Bender,
P. E. Bicyclic imidazoles as a novel class of cytokine bio-
synthesis inhibitors. Ann. N.Y. Acad. Sci. 1993, 696, 149.
3. (a) Lee, J. C.; Laydon, J. T.; McDonnell, P. C.; Gallagher,
T. F.; Kumar, S.; Green, D.; McNulty, D.; Blumenthal, M. J.;
Heys, J. R.; Landvatter, S. W.; Strickler, J. E.; McLaughlin,
M. M.; Siemens, I. R.; Fisher, S. M.; Livi, G. P.; White, J. R.;
Adams, J. L.; Young, P. R. A protein kinase involved in the
regulation of in¯ammatory cytokine biosynthesis. Nature
(London) 1994, 372, 736. (b) Han, J.; Lee, J.-D.; Bibbs, L.;
Ulevitch, R. J. A MAP kinase targeted by endotoxin and
hyperosmolarity in mammalian cells. Science 1994, 265, 808.
4. Hanson, G. J. Review, pulmonary-allergy, dermatological,
gastrointestinal and arthritis. Expert Opin. Ther. Pat. 1997, 7,
729.
Human CYP inhibition assay. Rapid inhibition assays
were undertaken to determine the inhibitory potency
(IC50) of the p38 compounds using Gentest recombinant
human CYPs. Probe substrates were incubated with the
expressed CYPs in the presence or absence of test com-
pound (0.4, 2, 10 and 50 mM). Gentest protocols were
followed except for a reduction in the incubation for
CYP2C9 assays (30 min).28
5. Astles, P. C.; Ashton, M. J.; Bridge, A. W.; Harris, N. V.;
Hart, T. W.; Parrott, D. P.; Porter, B.; Riddell, D.; Smith, C.;
Williams, R. J. Acyl-CoA:cholesterol O-acyltransferase
(ACAT) inhibitors. 2.2-(1,3-Dioxan-2-yl)-4,5-diphenyl-1H-
imidazoles as potent inhibitors of ACAT. J. Med. Chem. 1996,
39, 1423.
Guinea pig adrenotoxicity assay. Three guinea pigs were
dosed orally with compound at 100, 300 and 800 mg/kg/
day for 4 days. Animals were sacri®ced and adrenal
glands examined histologically for anomalies.
6. Badger, A. M.; Bradbeer, J. N.; Votta, B; Lee, J. C.;
Adams, J. L.; Griswold, D. E. Pharmacological pro®le of
SB203580, a selective inhibitor of cytokine suppressive binding
protein/p38 kinase, in animal models of arthritis, bone
resorption, endotoxic shock and immune function. J. Phar-
macol. Exp. Ther. 1996, 279, 1453.
7. Adams, J. L.; Boehm, J. C.; Kassis, S.; Gorycki, P. D.;
Webb, E. F.; Hall, R.; Sorenson, M.; Lee, J. C.; Ayrton, A.;
Griswold, D. E.; Gallagher, T. F. Pyrimidinylimidazole inhi-
bitors of CSBP/P38 kinase demonstrating decreased inhibition
of hepatic cytochrome P450 enzymes. Bioorg. Med. Chem.
Lett. 1998, 8, 3111.
8. Jackson, R. J.; Bolognese, B.; Hillegrass, L.; Kassis, S.;
Adams, J.; Griswold, D.; Winkler, J. D. Pharmacological
e€ects of SB 2200025, a selective inhibitor of P38 mitogen-
activated protein kinase, in angiogenesis and chronic in¯am-
matory disease models. J. Pharmacol. Exp. Ther. 1998, 284,
687.
Bacterialreverse mutation test (Ames test)
The potential of the compounds to induce point muta-
tions was evaluated in three Salmonella typhimurium
strains TA98, TA100 and TA102, both with and with-
out metabolic activation by liver homogenate from rats
pretreated with Aroclor (S9 mix), using plate incor-
poration method.29 The compound was considered
positive when it induces at least a 2-fold increase in the
number of revertant colonies.
9. Adams, J. L.; Gallagher, T. F.; Boehm, J. C.; Gum, R. J.;
Kassis, S.; Gorycki, P. D. p38 MAP kinase inhibitors: pro-
gress and possibilities. Presentation at the XVth International
Symposium on Medicinal Chemistry, Edinburgh, Scotland,
1998.
10. Denny, W. A. ATP site directed kinase inhibitors [meeting
report215ht ACS] . Idrugs 1998, 1, 10. Lewis, A. Intracellular
signaling and proteinase inhibition [meeting report 15th
EFMC]. Idrugs 1998, 1, 873.
11. de Laszlo, S. E.; Visco, D.; Agarwal, L. C.; Chin, J.;
Croft, G.; Forsyth, A.; Fletcher, D.; Frantz, B.; Hacker, C.;
Hanlon, W.; Harper, C.; Kostura, M.; Li, B.; Luell, S.; Mac-
Cross, M.; Mantlo, N.; O'Neill, E. A.; Orevillo, C.; Pang, M.;
Parsons, J.; Rolando, A.; Sahly, Y.; Sidler, K.; Widmer, W. R.;
O'Keefe, S. J. Pyrrols and other heterocycles as inhibitors of
p38 kinase. Bioorg. Med. Chem. Lett. 1998, 8, 2689.
In vitro micronucleus test in Chinese hamster ovary
(CHO) cells
The potential to induce chromosome damage was eval-
uated by measuring the increase in the number of
micronucleated cells. CHO-WBL cells were exposed for
3 h to the compound in the presence or in the absence of
metabolic activation by liver homogenate from rats pre-
treated with Aroclor (S9 mix). After treatment, cells were
grown for 20 h with 2 mg/mL cytochalasine B in order to
inhibit cytoplasm division without inhibiting nucleus
division. At the end of this 20-h period, cells were grown
for 1.5 h and then ®xed and stained. The compound was
considered positive if it induces at least a 2-fold increase
in the incidence of micronucleated cells.

554
I. M. McLay et al. / Bioorg. Med. Chem. 9 (2001) 537±554
12. Henrey, J. R.; Rupert, K. C.; Dodd, J. H.; Ignatius, J. T.;
Wadsworth, S. A.; Cavender, D. E.; Fahmy, B.; Olini, G. C.;
Davis, J. E.; Pellegrino-Gensey, J. L.; Scha€er, P. H.; Siekierka,
J. J. 6-Amino-2-(4-¯uorophenyl)-4-methoxy-3-(4-pyridyl)-1H-
pyrrolo[2,3-b]pyridine (RWJ 68354): A potent and selective
p38 kinase inhibitor. J. Med. Chem. 1998, 41, 4196.
13. Salituro, F. G.; Germann, U. A.; Wilson, K. P.; Bemis,
G. W.; Fox, T.; Su, M. S.-S. Inhibitors of p38 MAP kinase:
therapeutic intervention in cytokine-mediated disease. Curr.
Med. Chem. 1999, 6, 807.
14. (a) R & D Focus Drugs News, 17 May 1999. (b) Badger,
A. M.; Griswold, D. E.; Kapadia, R.; Blake, S.; Swift, B. A.;
Ho€man, S. J.; Stroup, G. B.; Webb, E.; Rieman, D. J.;
Gowen, M.; Boehm, J. C.; Adams, J. L.; Lee, J. C. Disease-
modifying activity of SB 242235, a selective inhibitor of p38
mitogen-activated protein kinase, in rat adjuvant-induced
arthritis. Arthritis Rheum. 2000, 43, 175.
15. (a) R & D Focus Drugs News, 15 November 1999. (b)
Structure disclosed in: Boehm, J. C.; Adams, J. L. New inhi-
bitors of p38 kinase. Expert Opin. Ther. Pat. 2000, 10, 25. (c)
Ferraccioli, G. F. VX-745 Vertex Pharmaceuticals. Curr. Opin.
Anti-In¯ammatory Immunomodulatory Invest. Drugs 2000, 2,
74.
16. Tong, L.; Pav, S.; White, D. M.; Rogers, S.; Crane, K. M.;
Cywin, C. L.; Brown, M. L.; Pargellis, C. A. A highly speci®c
inhibitor of human p38 MAP kinase binds in the ATP pocket.
Nat. Struct. Biol. 1997, 4, 311.
20. Short, B. G.; Apostoli, A. R.; Zimmerman, D. M.; Leo-
nard, T. B.; Schwartz, L. W. SK&F 105809 initiates and pro-
motes hepatic foci in rats. Proc. Amer. Assoc. Cancer Res.
1994, 35, 170.
21. Howard, M. O.; Schwartz, L. W.; Newton, J. F.; Qualls,
C. W., Jr.; Yodis, L. A. P.; Ventre, J. R. Comparative bio-
chemical and morphometric changes associated with induction
of the hepatic mixed function oxidase system in the rat. Tox-
icol-Pathol. 1991, 19, 115.
22. Miller, B. M.; Pujadas, E.; Gocke, E. Evaluation of the
micronucleus test in vitro using Chinese hamster cells: results
of four chemicals weakly positive in the in vivo micronucleus
test. Environ. Mol. Mutagen. 1995, 26, 240.
23. Ionnides, C.; Parke, D. V. Induction of cytochrome P4501
as an indicator of potential chemical carcinogenesis. Drug
Metab. Rev. 1993, 25, 485.
24. Roth, B. D. ACAT inhibitors: evolution from cholesterol-
absorption inhibitors to antiatherosclerotic agents. Drug Dis-
covery Today 1998, 3, 19.
25. Boehm, J. C.; Smietana, J. M.; Sorenson, M. E.; Gar-
igipati, R. S.; Gallagher, T. F.; Sheldrake, P. L.; Bradbeer, J.;
Badger, A. M.; Laydon, J. T.; Lee, J. C.; Hillegass, L. M.;
Griswold, D. E.; Breton, J. J.; Chabot-Fletcher, M. C.;
Adams, J. L. 1-Substituted 4-aryl-5-pyridinylimidazoles: A
new class of cytokine suppressive drugs with low 5-lipoxy-
genase and cyclooxygenase inhibitory potency. J. Med. Chem.
1996, 39, 3929.
17. Wilson, K. P.; McCa€rey, P. G.; Hsiao, K.; Pazhanisamy,
S.; Galullo, V.; Bemis, G. W.; Fitzgibbon, M. J.; Caron, P. R.;
Murcko, M. A.; Su, M. S. S. The structural basis for the spe-
ci®city of pyridinylimidazole inhibitors of p38 MAP kinase.
Chem. Biol. 1997, 4, 421.
18. Gum, R. J.; McLaughlin, M. M.; Kumar, S.; Wang, Z.;
Bower, M. J.; Lee, J. C.; Adams, J. L.; Livi, G. P.; Goldsmith,
E. J.; Young, P. R. Acquisition of sensitivity of stress-acti-
vated protein kinases to the p38 inhibitor, SB 203580, by
alteration of one or more amino acids within the ATP binding
pocket. J. Biol. Chem. 1998, 273, 15605.
26. Cook, D. C.; Lythgoe, D. J.; Thompson, D. M.; Wallace,
Paul A.; Walton, J. B. Chemical development and scale-up of
RPR200765A, a novel p38 anti-in¯ammatory candidate. Org.
Process Res. Dev. Submitted for publication.
27. Tzung, L. S.; Yuan, L. C. Synthesis of amino-containing
phosphines. The use of iminophosphorane as a protecting
group for primary amines. J. Org. Chem. 1992, 57, 6079.
28. Protocol can be obtained from the site: http://www.gen-
test.com.
29. Ames, B. N.; McCann, J.; Yamasaki, E. Methods for
detecting carciogens and mutagens with the salmonella/mam-
malian-microsome mutagenicity test. Mutat. Res. 1975, 31,
347.
19. Eyers, P. A.; Craxton, M.; Morrice, N.; Cohen, P.; Goe-
dert, M. Chem. Biol. 1998, 5, 321.